无乳链球菌诱导吉富罗非鱼分泌型免疫球蛋白M(sIgM)重链基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Secretory Form of Immunoglobulin M(sIgM)Heavy Chain Gene in GIFT Strain of Nile Tilapia(Oreochromis niloticus)Induced by Streptococcus agalactiae

作　　者：王培[1,2,3] 鲁义善[1,2,3] 王蓓[1,2,3] 汤菊芬[1,2,3] 蔡佳[1,2,3] 吴灶和[2,3,4] 简纪常[1,2,3] 

机构地区：[1]广东海洋大学水产学院,湛江524088 [2]广东省水产经济动物病原生物学及流行病学重点实验室,湛江524088 [3]广东省水产经济动物病害控制重点实验室,湛江524088 [4]仲恺农业工程学院,广州510225

出　　处：《生物技术通报》2014年第2期116-123,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(41240041);广东省科技计划(2012B020308010);广东省教育厅育苗项目(B12123)

摘　　要：以灭活的无乳链球菌(Streptococcus agalactiae)诱导后的吉富罗非鱼(Oreochromis niloticus)为材料,构建其头肾SMART cDNA文库,应用同源性克隆和RACE-PCR技术克隆到吉富罗非鱼(O.niloticus)分泌型免疫球蛋白M(sIgM)重链基因的全长cDNA序列并对其进行生物信息学分析。sIgM基因cDNA全长为1 921 bp,开放阅读框(ORF)为1 740 bp,5'端非编码区(5'-UTR)41 bp,3'端非编码区(3'-UTR)140 bp,编码579个氨基酸,N端有信号肽结构。预测分子量(MW)为64.26 kD,理论等电点(pI)为5.36。系统进化树分析显示,吉富罗非鱼(O.niloticus)sIgM与牙鲆(Paralichthys olivaceus)和军曹鱼(Rachycentron canadum)的亲缘关系较近。sIgM基因有4个恒定区。将吉富罗非鱼(O.niloticus)sIgM基因恒定区序列克隆到原核表达载体pET-28a(+)中,构建原核表达质粒pET28-sIgM,诱导表达后确定最优条件为37℃条件下,IPTG浓度为0.05 mmol/L时诱导4 h蛋白表达量最大。纯化蛋白后经Western blot分析,sIgM融合蛋白与鼠抗His-Tag发生特异结合,表明目的蛋白成功表达。GIFT strain of Nile tilapia（Oreochromis niloticus）, induced by inactivated Streptococcus agalactiae, was used to construct the head kidney cDNA library. The full-length sIgM cDNA of Nile Tilapia was cloned using homological cloning and rapid amplification of cDNA ends（RACE）methods. Results showed the full-length of sIgM cDNA was 1 921 bp, containing a 5＇ untranslated region（5＇-UTR）of 41 bp, a 3＇-UTR of 140 bp and an open reading frame of 1 740 bp encoding 579 amino acids with an estimated molecular weight of 64.26 kD and an estimated isoelectric point of 5.36. In the N-terminal of sIgM exists a signal peptide structure. The phylogenetic trees constructed showed that sIgM of Nile tilapia shared the closest relationship with the corresponding proteins of Paralichthys olivaceus and Rachycentron canadum. The sIgM had four CH domains. The constant segment of sIgM gene was amplified and inserted into the pET-28a（＋）vector to construct the prokaryotic expression plasmid pET28a-sIgM. The optimal expression condition was set as 0.05 mmol/ L IPTG, induced temperature 37℃, and induced time 4 hours.The recombinant sIgM fusion proteins was purified by His TrapTM HP column, and then identified by western blot using His-Tag Mouse mAb.

关 键 词：吉富罗非鱼 免疫球蛋白M 基因克隆 原核表达 蛋白免疫印迹 

分 类 号：S943[农业科学—水产养殖]